Cryopreservation of Embryos of Farm Animals

Abstract

This review contains two parts. The first part is devoted to the significant steps in cryopreservation of mammalian embryos with emphasis on cattle and sheep that serve as models of reference. These steps are: (1) shortening of cooling and warming processes; (2) addition and dilution of cryoprotectant in one step; (3) introduction of plastic straw as a freezing and dilution container; (4) the choice of ethylene glycol as the quite universal cryoprotectant because of its low toxicity and high permeability; (5) vitrification, a cryopreservation method which enable passage from the liquid to the solid state by extreme elevation of viscosity due to high concentration of cryoprotectants and very rapid cooling. There are several vitrification solutions which contain dimethyl sulphoxide, glycerol, ethylene glycol, or a mixture of them, as basic cryoprotectants. The second part considers some factors affecting the efficiency of cryopreservation concerning (i) the origin of embryos and (ii) the stage of development and species. The origin of embryos (in vivo versus in vitro): in vitro embryos show a chilling and freezing sensitivity associated with their lipid content which can be modified by the culture conditions. Both conventional freezing and vitrification have been used and it seems that vitrification is more adapted to in vitro embryos when some modifications of initial protocols are carried out, particularly the rate of cooling. Thus considerable progress has been achieved by using the open pulled straw method of Vajta which enables the use of a minimum volume of freezing medium (0.5 microl) and a very high cooling rate that permits rapid traversal of the damaging temperature zone, corresponding to chilling sensitivity. The stage of development and species: not only are there differences between species at the same stage of development but in the same species all stages of development do not survive equally under the same freezing protocol. In cattle for example, oocytes and early stages of development in vivo or in vitro do not survive whereas compacted morulae and blastocysts survive very well. In the pig hatched blastocysts survive better than the other stages. Horse embryos have special characteristics that pose problems for successful freezing. In conclusion, a lot of work remains to be done to define fundamental characteristics of embryos of certain species (pig, horse) and of embryos of some stages or of oocytes.

Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol

“…Previous work with mature caprine oocytes indicated that the permeability of GLY was highly dependent on temperature, and at 20 °C [4] and 4 °C [16], this cryoprotectant has lower permeability than EG. Newton et al [18] compared the efficiency of four cryoprotectants (DMSO, propanediol, EG and GLY) in the cryopreservation of human ovarian tissue; they showed that EG had better results because it penetrated more quickly in the tissue, due probably to its lower molecular weight [50]. Based on the literature [12] and [16], EG was more effective at protecting cells during cryopreservation than GLY, perhaps due to its low toxicity and efficient permeability [51].…”

Criopreservação De Folículos Pré-Antrais Caninos Com Glicerol E Etilenoglicol

“…O glicerol possui peso molecular superior ao etilenoglicol (92,10 vs. 62,07 dáltons;Massip, 2001), esta característica confere ao glicerol baixa permeabilidade de membrana, o que leva a severos danos citoplasmáticos (Széll e Shelton, 1986). Diversos estudos têm demonstrado que a exposição do tecido ovariano ou de folículos pré-antrais ao glicerol, resulta em uma redução substancial na porcentagem de folículos viáveis em caprinos (Rodrigues et al, 2004) e ovinos (Santos et al, 2006).…”

Cryopreservation of Sheep Produced Embryos – Current and Future Perspectives

“…In fact, vitrification is based on embryo manipula-tion into different carrier tools applied to minimize the volume and to submerge the sample quickly into liquid nitrogen, allowing an ultrafast freezing speed, which avoids ice crystal formation, thus eliminating its deleterious effects in the cell [157]. This technique combines the use of small volumes with high concentration of two or more cryoprotectants [158] and it can be more adapted to IVP embryos [149,159,160].…”